type-A α-toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a 4·6 gradient. Milligram yields of highly purified α-toxin with 10 to 15% recovery of activity were obtained in single fractions.

Two forms of α-toxin were identified by electrofocusing. The major peak of activity possessed a of 5·49±0·06(α) and the minor, a of 5·25(α). The former was free from detectable collagenase 4·54), hyaluronidase 4·73), θ-toxin 6·56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with α in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of α.

Fractions α and α both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of α-toxin in the presence of 6m urea resulted in the detection of only one component, α, with a identical to α. It also possessed all three biological activities of α-toxin. Removal of the urea and refocusing was accompanied by the reappearance of α. The occurrence and formation of α could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that α and α could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.


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